ABSTRACT There is a significant gap in understanding how GM-CSF directs macrophage specification following pulmonary macrophage transplantation (PMT). This is an important problem, because, without this crucial information, the preclinical data will not adequately demonstrate PMT safety sufficient to gain approval to test PMT in humans. The long-term goal is to develop PMT as therapy for patients with hereditary pulmonary alveolar proteinosis (hPAP) aimed at restoring alveolar macrophage (AM) function. The objective here is to identify mechanism(s) directing AM specification during PMT. The central Hypothesis is that AM specification is determined primarily by factors in the alveolar microenvironment including 1) GM-CSF, which renders AM-specific DNA regulatory elements accessible; and 2) surfactant phospholipid-derived fatty acids, which activate PPAR? to bind newly accessible DNA and switch AMs from LXR-driven to PPAR?-driven specification (and cholesterol metabolism). Our rationale is that by demonstrating GM-CSF directs macrophages to adopt a normal AM transcription profile at single cell resolution (which `bulk' studies failed to show) will be crucial to establish PMT is safe. Guided by our strong preliminary data, this hypothesis will be tested by pursuing three specific aims to determine 1) the relationship between donor cell plasticity and therapeutic efficacy; 2) temporal dynamics of macrophage engraftment, specification, and fate after PMT; and 3) cis-regulatory architecture governing AM specification. In aim 1, myeloid cells of various developmental stages will be administered by PMT to Csf2raKO mice to deter- mine the plasticity of cells capable of conferring therapeutic benefit. In aim 2, PMT of normal hematopoietic stem/progenitor cell (HSPC) donors in Csf2raKO (or normal) mice with/without M-CSF inhibition will be done to determine donor survival, proliferation, clonal expansion, phenotype and function. Cell population dynamics will be tracked temporally by single-cell RNA-seq using Monocle. In aim 3, mouse or human HSPCs will be cultured with/without GM-CSF and surfactant or DPPC and gene regulatory dynamics will be measured by single-cell RNA-seq using Monocle to determine differentiation `trajectories'. DNA elements regulating cell population dynamics will be identified by measuring chromatin accessibility by sci-ATAC-seq and using Cicero to link DNA regulatory elements to the target genes they regulate. The proposed research is innovative because it supports the development of a new therapy for hPAP based on restoring AM function rather than physically removing surfactant (the current approach), and because it uses novel methods, software and statistical tools to identify elements directing AM specification as `upstream regulators' or `downstream' targets, which has not been possible with methods available previously. The proposed research is significant because it will identify the alveolar determinants and transcriptional mechanism(s) regulating AM specification during PMT, support the development of PMT as an effective, disease-specific cell therapy for children with hPAP, and provide a foundation for developing novel macrophage-based therapies for other lung diseases.